Fine structure of single fibres of human skeletal muscle

Summary Individual muscle fibres were separated from freeze-dried needle biopsies and classed as type I or type II fibres according to their myofibrillar ATP-ase. Portions of the same fibres were processed for electron microscopy and their fine structure examined. Type I fibres were found to have thicker Z-bands and more mitochondria and lipid droplets than the type II fibres.     

Fine structural details of human muscle fibres after fibre type specific glycogen depletion

Summary Type 1 and Type 2 fibres of skeletal muscle (human m. vastus lateralis), selectively depleted of glycogen by sustained submaximal muscular exercise (running 30 km), were identified at light and electron microscopical level by examination of thin and ultra-thin serial sections treated particularly for visualization of glycogen. Averaged images, obtained by lateral smearing of depleted fibres (Type 1) exhibited five clearly visible cross-bridges in the M-band and had broad Z-bands. Nondepleted fibres (Type 2) showed either three central strong and two weak outer lines in the M-band and intermediate Z-bands (Type 2A), or only three central strong lines in the M-band and narrow Z-bands (Type 2B). The depleted fibres had no subsarcolemmal accumulation of glycogen particles and practically no intermyofibrillar particles. The remaining particles were small in size and seemed almost rudimentary. In nonexercised individuals, a peculiar distribution of individual glycogen particles in the I-band and A-band was found. This distribution was accounted by the structural arrangement of the myofibrillar material.     

Purinergic receptors expressed in human skeletal muscle fibres

Purinergic receptors are present in most tissues and thought to be involved in various signalling pathways, including neural signalling, cell metabolism and local regulation of the microcirculation in skeletal muscles. The present study aims to determine the distribution and intracellular content of purinergic receptors in skeletal muscle fibres in patients with type 2 diabetes and age-matched controls. Muscle biopsies from vastus lateralis were obtained from six type 2 diabetic patients and seven age-matched controls. Purinergic receptors were analysed using light and confocal microscopy in immunolabelled transverse sections of muscle biopsies. The receptors P2Y(4), P2Y(11) and likely P2X(1) were present intracellularly or in the plasma membrane of muscle fibres and were thus selected for further detailed morphological analysis. P2X(1) receptors were expressed in intracellular vesicles and sarcolemma. P2Y(4) receptors were present in sarcolemma. P2Y(11) receptors were abundantly and diffusely expressed intracellularly and were more explicitly expressed in type I than in type II fibres, whereas P2X(1) and P2Y(4) showed no fibre-type specificity. Both diabetic patients and healthy controls showed similar distribution of receptors. The current study demonstrates that purinergic receptors are located intracellularly in human skeletal muscle fibres. The similar cellular localization of receptors in healthy and diabetic subjects suggests that diabetes is not associated with an altered distribution of purinergic receptors in skeletal muscle fibres. We speculate that the intracellular localization of purinergic receptors may reflect a role in regulation of muscle metabolism; further studies are nevertheless needed to determine the function of the purinergic system in skeletal muscle cells.     

Fine structure and metabolism of multiply innervated fast muscle fibres in teleost fish

Summary Both the fast and slow muscle fibres of advanced teleost fish are multiply innervated. The fraction of slow-fibre volume occupied by mitochondria is 31.3%, 25.5% and 24.6%, respectively, for the myotomal muscles of brook trout (Salvelinus fontinalis), crucian carp (Carassius carassius), and plaice (Pleuronectes platessa), respectively. The corresponding figures for the fast muscles of these species are 9.3%, 4.6% and 2.0%, respectively. Cytochrome-oxidase and citrate-synthetase activities in the fast muscles of 9 species of teleost range from 0.20–0.93 moles substrate utilised, g wet weight muscle^-1 min^-1 (at 15° C) or around 4–17% of that of the corresponding slow fibres. Ultrastructural analyses reveal a marked heterogeneity within the fast-fibre population. For example, the fraction of fibres with <1% or >10% mitochondria is 0,4,42% and 36, 12 and 0%, respectively, for trout, carp and plaice. In general, small fibres (<500 m²) have the highest and large fibres (>1,500 m²) the lowest mitochondrial densities. The complexity of mitochondrial cristae is reduced in fast compared to slow fibres.Hexokinase activities range from 0.4–2.5 in slow and from 0.08–0.7 moles, g wet weight^-1 min^-1 in fast muscles, indicating a wide variation in their capacity for aerobic glucose utilisation. Phosphofructokinase activities are 1.2 to 3.6 times higher in fast than slow muscles indicating a greater glycolytic potential. Lactate dehydrogenase activities are not correlated with either the predicted anaerobic scopes for activity or the anoxic tolerances of the species studied. The results indicate a considerable variation in the aerobic capacities and principal fuels supporting activity among the fast muscles of different species. Brook trout and crucian carp are known to recruit fast fibres at low swimming speeds. For these species the aerobic potential of the fast muscle is probably sufficient to meet the energy requirements of slow swimming.     

Collagen of slow twitch and fast twitch muscle fibres in different types of rat skeletal muscle

The appearance of collagen around individual fast twitch (FT) and slow twitch (ST) muscle fibres was investigated in skeletal muscles with different contractile properties using endurance trained and untrained rats as experimental animals. The collagenous connective tissue was analyzed by measuring hydroxyproline biochemically and by staining collagenous material histochemically in M. soleus (MS), M. rectus femoris (MRF), and M. gastrocnemius (MG). The concentration of hydroxyproline in the ST fibres dissected from MS (2.72 +/- 0.35 micrograms X mg-1 d.w.) was significantly higher than that of the FT fibres dissected from MRF (1.52 +/- 0.33 micrograms X mg-1 d.w.). Similarly, the concentration of hydroxyproline was higher in ST (2.54 +/- 0.51 micrograms X mg-1 d.w.) than in FT fibres (1.60 +/- 0.43 micrograms X mg-1 d.w.), when the fibres were dissected from the same muscle, MG. Histochemical staining of collagenous material agreed with the biochemical evidence that MS and the slow twitch area of MG are more collagenous than MRF and the fast twitch area of MG both at the level of perimysium and endomysium. The variables were not affected by endurance training. When discussing the role of collagen in the function of skeletal muscle it is suggested that the different functional demands of different skeletal muscles are also reflected in the structure of intramuscular connective tissue, even at the level of endomysial collagen. It is supposed that the known differences in the elastic properties of fast tetanic muscle compared to slow tonic muscle as, e.g., the higher compliance of fast muscle could at least partly be explained in terms of the amount, type, and structure of intramuscular collagen.     

Insect visceral muscle. Fine structure of the proctodeal muscle fibres

The fine structure of the longitudinal muscle fibres of the cockroach proctodeum was investigated by electron microscopy. The fibre is separated incompletely into fibrils, the resting sarcomere length is variable: about 5·8 to 7·3 μm, and the A- and I-bandings are not always clear in longitudinal sections. The ratio of thin and thick filaments at the overlapped region is about 4·1:1 when relaxed, and about 9·8:1 when fully contracted. The myofilament array is not well organized.The previously observed prolonged time course of muscle contraction seems to correlate with the present observations on the poorly developed sarcoplasmic reticulum and irregular distribution of transverse tubules. The Z-bands are irregularly aligned and discontinuous in longitudinal sections. The Z-band structure was studied in relation to the supercontractility. It was found that at maximal isotonic contraction (about 25 per cent rest length) the myofilaments pass through the expanded Z-regions.     

Age-related changes in the reducible cross-links on connectin from human skeletal muscle.

After NaB³H₄-reduction of connectin from human skeletal muscle, the changes in the amounts of the reducible cross-links and specific radioactivity of this elastic protein were followed throughout the whole life-span from embryo to old age. The reducible cross-links, aldimine forms of lysinonorleucine and histidino-hydroxymerodesmosine, and unidentified reducible compounds, which were assumed to be cross-linking amino acids, were found to remarkably decrease with age. A progressive decrease in the incorporation of tritium into the reducible compounds was also observed. We conclude that the conversion of the reducible cross-links derived from lysine and hydroxylysine aldehydes to non-reducible compounds is an essential step in the maturation of connectin fibrils, similar to collagen fibrils.     

Age-related changes in ATP-producing pathways in human skeletal muscle in vivo.

Energy for muscle contractions is supplied by ATP generated from 1) the net hydrolysis of phosphocreatine (PCr) through the creatine kinase reaction, 2) oxidative phosphorylation, and 3) anaerobic glycolysis. The effect of old age on these pathways is unclear. The purpose of this study was to examine whether age may affect ATP synthesis rates from these pathways during maximal voluntary isometric contractions (MVIC). Phosphorus magnetic resonance spectroscopy was used to assess high-energy phosphate metabolite concentrations in skeletal muscle of eight young (20-35 yr) and eight older (65-80 yr) men. Oxidative capacity was assessed from PCr recovery after a 16-s MVIC. We determined the contribution of each pathway to total ATP synthesis during a 60-s MVIC. Oxidative capacity was similar across age groups. Similar rates of ATP synthesis from PCr hydrolysis and oxidative phosphorylation were observed in young and older men during the 60-s MVIC. Glycolytic flux was higher in young than older men during the 60-s contraction (P < 0.001). When expressed relative to the overall ATP synthesis rate, older men relied on oxidative phosphorylation more than young men (P = 0.014) and derived a smaller proportion of ATP from anaerobic glycolysis (P < 0.001). These data demonstrate that although oxidative capacity was unaltered with age, peak glycolytic flux and overall ATP production from anaerobic glycolysis were lower in older men during a high-intensity contraction. Whether this represents an age-related limitation in glycolytic metabolism or a preferential reliance on oxidative ATP production remains to be determined.     

Nucleotide binding induces global and local structural changes of myosin head in muscle fibres.

Thermal stability and internal dynamics of myosin heads in fiber bundles from rabbit psoas muscle has been studied by electron paramagnetic resonance (EPR) spectroscopy and differential scanning calorimetry (DSC). Using ADP, ATP and orthovanadate (V(i)), three intermediate states of the ATP hydrolysis cycle were simulated in glycerinated muscle fibers. DSC transitions contained three overlapping endotherms in each state. Deconvolution showed that the transition temperature of 58.4 degrees C was almost independent of the intermediate state of myosin, while nucleotide binding shifted the melting temperatures of 54.0 and 62.3 degrees C, and changed the enthalpies. These changes suggest global rearrangements of the internal structure in myosin head. In the presence of ADP and ADP plus V(i), the conventional EPR spectra showed changes in the ordering of the probe molecules, suggesting local conformational and motional changes in the internal structure of myosin heads. Saturation transfer EPR measurements reported increased rotational mobility of spin labels in the presence of ATP plus orthovanadate corresponding to a weakly binding state of myosin to actin.     

Histochemical heterogeneity of intrafusal muscle fibres in slow and fast skeletal muscles of the rat

Summary Intrafusal muscle fibres of the slow soleus (Sol) and fast vastus lateralis (VL) muscles of the rat were studied histochemically. Serial transverse sections were incubated for the localization of succinate dehydrogenase (SDH), alpha glycerophosphate dehydrogenase (GPD) and adenosine triphosphatase (ATPase). The latter was examined further after preincubation in acidic solution held at either low or room temperature (R_T). The bag₂ intrafusal fibres in both muscles displayed high regular and acid stable ATPase, but low SDH and GPD activities. Bag₁ intrafusal fibres showed low to moderate regular ATPase, a regional heterogeneity after R_T acid preincubation (low activity in juxtaequatorial and high in polar zones), moderate SDH, but low GPD reactions. In both muscles the chain fibres usually exhibited high ATPase for both regular and cold acid preincubated reactions, but usually low activity after R_T acid preincubation; they had high SDH but variable GPD activities. In Sol muscle, however, approximately 25% of spindles contained chain fibres that showed high acid-stable ATPase reaction after both cold and R_T acid preincubation. In contrast, chain fibres in some VL spindles had a characteristically low ATPase reaction even after cold acid preincubation. This study, therefore, has delineated the existence of an inherent heterogeneity among chain fibres (with respect to their histochemical reactions) in muscle spindles located within slow and fast muscles and also between those found within populations of either Sol or VL muscle spindles.     

Light diffraction study of single skeletal muscle fibres.

Light diffraction patterns from isolated frog semitendinosus muscle fibers were examined. When transilluminated by laser light, the muscle striations produce a diffraction pattern consisting of a series of lines that are projected as points onto an optical detector by a lens system. Diffraction data may be sequentially stored every 18 ms for later processing by digital computer systems. First- and second-order diffraction line intensities were examined from intact, chemically skinned, and glycerinated single fibers. The diffraction line intensities demonstrated a strong length dependence upon passive stretch from reference length to 3.6 micrometer. The first-order intensity linearly increased an average of 15-fold over the range examined. The magnitude of the second order intensity was less than the first order and showed an exponential rise with increasing length. Both first- and second-order intensities decreased upon muscle activation. Data from chemically skinned and glycerinated single fibers were not significantly different from intact fibers, indicating that the membrane structure has little effect upon the diffraction phenomenon in muscle. Theoretical model systems are examined in an attempt to find the basis of these results. Neither an analysis based on a diffraction grating with variable spacing nor the unit cell model of Fujime provides an explanation for the observed length dependency of intensity. Though the origin of the intensity decrease upon stimulation is not known, we have suggested that it could result from lateral misalignment of myofibrils and can occur upon activation.     

Temperature dependence of the inhibitory effects of orthovanadate on shortening velocity in fast skeletal muscle.

We have investigated the effects of the orthophosphate (P(i)) analog orthovanadate (Vi) on maximum shortening velocity (Vmax) in activated, chemically skinned, vertebrate skeletal muscle fibers. Using new "temperature-jump" protocols, reproducible data can be obtained from activated fibers at high temperatures, and we have examined the effect of increased [Vi] on Vmax for temperatures in the range 5-30 degrees C. We find that for temperatures < or = 20 degrees C, increasing [Vi] inhibits Vmax; for temperatures > or = 25 degrees C, increasing [Vi] does not inhibit Vmax. Attached cross-bridges bound to Vi are thought to be an analog of the weakly bound actin-myosin.ADP-P(i) state. The data suggest that the weakly bound Vi state can inhibit velocity at low temperature, but not at high temperature, with the transition occurring over a narrow temperature range of < 5 degrees C. This suggests a highly cooperative interaction. The data also define a Q10 for Vmax of 2.1 for chemically skinned rabbit psoas fibers over the temperature range of 5-30 degrees C.     

Evidence for a substrate regulation of triglyceride lipolysis in human skeletal muscle

Glycerol release from the human forearm which is generally used as a semiquantitative index of intramuscular lipolysis was studied under different hormonal influence and substrate supply in healthy volunteers and juvenile diabetics using the forearm technique. Acute insulin deficiency in juvenile diabetics failed stimulating the rate of muscular lipolysis since the rates of glycerol release in normals and diabetics were the same. In addition, in normal volunteers high physiological levels of insulin caused by an intraarterial infusion of the hormone exhibited no effect on the glycerol release from deep forearm tissue. Similarly, an intraarterial infusion of metaproterenol did not accelerate muscular glycerol release in normal man. However, in juvenile diabetics in acute insulin deficiency the same dose of the catecholamine increased the rate of muscular glycerol production. Elevated substrate supply during intravenous infusion of glucose or fructose yielded increased uptake of glucose and fructose into the deep forearm tissue and thereby promptly blocked muscular glycerol release in normal volunteers and in juvenile diabetics. These findings suggest that the rate of lipolysis in muscle tissue is not primarily under the control of hormones but rather by substrate supply.     

Post-mortem changes in skeletal muscle connectin.

Changes in connectin and elasticity of skeletal muscle were determined during post-mortem ageing. The amount of connectin decreased with increasing time of post-mortem storage whereas the rate of the decrease depended on the source of muscles. The loss in elasticity of muscle coincided well with the decrease in connectin contents. Electron microscopically, a network structure between the Z discs vanished when the amount of connectin fell to zero. We have concluded that the continuous net structure of connectin is responsible for about 30% of the total elasticity of living skeletal muscle and its degradtaion is responsible for post-mortem tenderization of meat.